Manipulating droplet size

ABSTRACT

The invention generally relates to methods and systems for manipulating droplet size. In certain aspects, the invention provides methods for manipulating droplet size that include forming droplets of aqueous fluid surrounded by an immiscible carrier fluid, and manipulating droplet size during the forming step by adjusting pressure exerted on the aqueous fluid or the carrier fluid.

RELATED APPLICATION

The present application claims benefit of and priority to U.S.provisional application Ser. No. 61/509,837, filed Jul. 20, 2011, thecontent of which is incorporated by reference herein in its entirety.

FIELD OF THE INVENTION

The invention generally relates to methods and systems for manipulatingfluidic droplet size.

BACKGROUND

The ability to precisely manipulate fluidic streams enhances the use andeffectiveness of microfluidic devices. Typically, networks of smallchannels provide a flexible platform for manipulation of small amountsof fluids. Certain microfluidic devices utilize aqueous droplets in animmiscible carrier fluid. The droplets provide a well-defined,encapsulated microenvironment that eliminates cross contamination andchanges in concentration due to diffusion or surface interactions.

Microfluidic devices for performing biological, chemical, and diagnosticassays generally include at least one substrate containing one or moteetched or molded channels. The channels are generally arranged to formindividual fluid circuits, each circuit including a sample fluidchannel, an immiscible carrier fluid channel, and an outlet channel. Thechannels of each circuit may be configured such that they meet at ajunction so that droplets of aqueous fluid surrounded by carrier fluidare formed at the junction and flow into the outlet channel. In somecases, the outlet channel of each circuit is connected to a main channelthat receives all of the droplets from the different fluidic circuitsand flows them to an analysis module. In other cases, the outletchannels connect to exit ports to carry the droplets to a collectionvessel.

Since each fluidic circuit may have different samples, and becausedifferent compositions (e.g., concentration and/or length of nucleicacid) from different samples affect how droplets form, droplets ofdifferent sizes may be produced by each circuit. A problem with dropletsof different sizes flowing through the same channel is that the dropletstravel at different velocities. Droplets traveling at differentvelocities may cause unwanted collisions or unwanted coalescence ofdroplets in the channel. Thus, it is important that individual fluidiccircuits produce droplets of uniform size so that the droplets travel atthe same velocity in the channel and do not collide or coalesce in anunwanted manner.

Droplets are typically generated one at a time at a junction between anaqueous fluid and an immiscible carrier fluid. Droplet volume andfrequency (the number of droplet generated per unit time) are determinedby geometrical factors such as the cross-sectional area of the channelsat the junction and the fluidic properties such as the fluid viscositiesand surface tensions as well as the infusion rates of the aqueous andcarrier fluids. To control the volume of the aqueous droplet, within arange, droplet volume can be adjusted by tuning the oil infusion ratethrough the junction. This is readily achieved with a pressure regulatoron the carrier fluid stream. In some cases it is desirable to havemultiple junctions operating as separate circuits to generate dropletsand have independent control over the oil infusion rates through eachcircuit. This is readily achieved by using separate pressure regulatorsfor each aqueous stream and each carrier fluid stream. A simpler andlower cost system would have a single carrier oil source at a singlepressure providing a flow of carrier oil through each system. Theproblem with such a system is that in adjusting the pressure to regulatethe flow of carrier oil in one circuit the carrier oil in all circuitswould be effected and independent control over droplet volume would becompromised. Thus, it is important to have a means whereby at a fixedcarrier oil pressure the flow of carrier oil in each of the circuits canbe independently controlled to regulate droplet volume.

SUMMARY

The invention generally relates to methods and systems for manipulatingdroplet size. The invention recognizes that in a fluidic circuit,changing the pressure exerted on the aqueous phase changes the flow rateof the immiscible carrier fluid. Changing the flow rate of theimmiscible fluid manipulates the size of the droplet. Thus, adjustingpressure, which changes flow rate, adjusts droplet size. Pressureadjustments may be made independent of one another such that thepressure exerted on the aqueous phase in individual fluidic circuits canbe adjusted to produce droplets of uniform size from the differentfluidic circuits. In this manner, droplets produced from differentfluidic circuits travel at the same velocity in a main channel and donot collide or coalesce in an unwanted manner.

In certain aspects, the invention provides methods for manipulatingdroplet size that involve forming droplets of aqueous fluid surroundedby an immiscible carrier fluid, and manipulating droplet size during theforming step by adjusting pressure exerted on the aqueous fluid or thecarrier fluid. Methods of the invention involve forming a sampledroplet. Any technique known in the art for forming sample droplets maybe used with methods of the invention. An exemplary method involvesflowing a stream of sample fluid so that the sample stream intersectstwo opposing streams of flowing carrier fluid. The carrier fluid isimmiscible with the sample fluid. Intersection of the sample fluid withthe two opposing streams of flowing earner fluid results in partitioningof the sample fluid into individual sample droplets. The carrier fluidmay be any fluid that is immiscible with the sample fluid. An exemplarycarrier fluid is oil. In certain embodiments, the carrier fluid includesa surfactant, such as a fluorosurfactant.

Methods of the invention may be conducted in microfluidic channels. Assuch, in certain embodiments, methods of the invention may furtherinvolve flowing the droplet channels and under microfluidic control.Methods of the invention further involve measuring the size of agenerated droplet. Any method known in the art may be used to measuredroplet size. Preferable methods involve realtime image analysis of thedroplets, which allows for a feedback loop to be created so that dropletsize may be adjusted in real-time. In certain embodiments, measuring thedroplet size is accomplished by taking an image of the droplet andmeasuring a midpoint of an outline of the droplet image, as opposed tomeasuring an inside or an outside of the droplet.

Another aspect of the invention provides methods for forming droplets ofa target volume that include flowing an aqueous fluid through a firstchannel, flowing an immiscible carrier fluid through a second channel,forming an aqueous droplet surrounded by the carrier fluid, andadjusting resistance in the first or second channels during the formingstep to adjust volume of the droplets, thereby forming droplets of atarget volume.

Another aspect of the invention provides methods for formingsubstantially uniform droplets that involve flowing a plurality ofdifferent aqueous fluids through a plurality of different channels,flowing an immiscible carrier fluid through a carrier fluid channel,forming substantially uniform droplets of the different aqueous fluids,each droplet being surrounded by the carrier fluid, by independentlyadjusting resistance in the different channels.

Another aspect of the invention provides microfluidic chips that includea substrate, and a plurality of channels, in which at least two of thechannels include pressure regulators, the pressure regulators beingindependently controllable. Generally, the plurality of channels includeat least one aqueous fluid channel, at least one immiscible carrierfluid channel, at least one outlet channel, and a main channel. Incertain embodiments, the channels are configured to form microfluidiccircuits, each circuit including an aqueous fluid channel, a carrierfluid channel, and an outlet channel. The channels of each circuit meetat a junction such that droplets of aqueous fluid surrounded by carrierfluid are formed at the junction and flow into the outlet channel. Eachoutlet channel of each circuit is connected to the main channel. Thechannels may be etched or molded into the substrate. The channels may beopen channels or enclosed channels. Droplets may be collected in avessel on the device or off of the device.

Another aspect of the invention provides droplet systems that include amicrofluidic chip that include a substrate, and a plurality of channels,in which at least two of the channels include pressure regulators, thepressure regulators being independently controllable; and a pressuresource coupled to the chip.

Other aspects and advantages of the invention are provided in thefollowing description and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a drawing showing a device for droplet formation.

FIG. 2 is a drawing showing a device for droplet formation.

FIG. 3 is a graph showing droplet size sensitivity to changes in aqueousflow rate when using positive displacement pumping.

FIG. 4 is a graph showing droplet size sensitivity to changes in aqueousflow rate when using pressure driven pumping.

FIG. 5 shows a diagram of a single fluidic circuit.

FIG. 6 is a drawing illustrating that the same volume drop is subject toextreme changes in the lighting but the midpoint is always the same.From left to right, the intensity of the lighting decreases but themidpoint of the outline is always the same.

FIGS. 7A-C provides three graphs that demonstrate the differences in thedroplet measuring techniques, and the projected area required to produce5 pL drops when using the inside, outside and midpoint of a dropletimage.

FIG. 8 is a schematic illustrating measurement of droplet size using themidpoint technique described herein.

FIG. 9 is a schematic diagram showing a micro fluidic interconnect asdescribed in the Specification, containing a plurality of aqueous fluidports and an immiscible fluid port for use in methods of the invention.

FIG. 10 is a schematic diagram showing an apparatus as described in theSpecification showing the microfluidic interconnect shown in FIG. 9 witha manifold overlay and immiscible fluid storage.

FIG. 11 is a schematic diagram showing the relationship between themicrofluidic interconnect of FIG. 9 with a microfluidic chip for use inmethods of the invention.

DETAILED DESCRIPTION

The invention generally relates to methods and systems for manipulatingdroplet size. In certain aspects, the invention provides methods formanipulating droplet size that involve forming droplets of aqueous fluidsurrounded by an immiscible carrier fluid, and manipulating droplet sizeduring the forming step by adjusting pressure exerted on the aqueousfluid or the carrier fluid.

Droplet Formation

Methods of the invention involve forming sample droplets. In certainembodiments, the droplets include nucleic acid from different samples.In particular embodiments, each droplet includes a single nucleic acidtemplate, a single protein molecule or single cell. The droplets areaqueous droplets that are surrounded by an immiscible carrier fluid.Methods of forming such droplets are shown for example in Link et al.(U.S. patent application numbers 2008/0014589, 08/0003142, and2010/0137163). Stone et al. (U.S. Pat. No. 7,708,949 and U.S. patentapplication number 2010/0172803), Anderson et al. (U.S. Pat. No.7,041,481 and which reissued as RE41,780) and European publicationnumber EP2047910 to Raindance Technologies Inc. The content of each ofwhich is incorporated by reference herein in its entirety.

FIG. 1 shows an exemplary embodiment of a device 100 for dropletformation. Device 100 includes an inlet channel 101, and outlet channel102, and two carrier fluid channels 103 and 104. Channels 101, 102, 103,and 104 meet at a junction 105. Inlet channel 101 flows sample fluid tothe junction 105. Carrier fluid channels 103 and 104 flow a carrierfluid that is immiscible with the sample fluid to the junction 105.Inlet channel 101 narrows at its distal portion wherein it connects tojunction 105 (See FIG. 2). Inlet channel 101 is oriented to beperpendicular to carrier fluid channels 103 and 104. Droplets are formedas sample fluid flows from inlet channel 101 to junction 105, where thesample fluid interacts with flowing carrier fluid provided to thejunction 105 by carrier fluid channels 103 and 104. Outlet channel 102receives the droplets of sample fluid surrounded by carrier fluid.

The sample fluid is typically an aqueous buffer solution, such asultrapure water (e.g., 18 mega-ohm resistivity, obtained, for example bycolumn chromatography), 10 mM Tris HCl and 1 mM EDTA (TE) buffer,phosphate buffer saline (PBS) or acetate buffer. Any liquid or bufferthat is physiologically compatible with enzymes can be used. The carrierfluid is one that is immiscible with the sample fluid. The carrier fluidcan be a non-polar solvent, decane (e g., tetradecane or hexadecane),fluorocarbon oil, silicone oil or another oil (for example, mineraloil).

In certain embodiments, the carrier fluid contains one or moreadditives, such as agents which reduce surface tensions (surfactants).Surfactants can include Tween, Span, fluorosurfactants, and other agentsthat are soluble in oil relative to water. In some applications,performance is improved by adding a second surfactant to the samplefluid. Surfactants can aid in controlling or optimizing droplet size,flow and uniformity, for example by reducing the shear force needed toextrude or inject droplets into an intersecting channel. This can affectdroplet volume and periodicity, or the rate or frequency at whichdroplets break off into an intersecting channel. Furthermore, thesurfactant can serve to stabilize aqueous emulsions in fluorinated oilsfrom coalescing.

In certain embodiments, the droplets may be coated with a surfactant.Preferred surfactants that may be added to the carrier fluid include,but are not limited to, surfactants such as sorbitan-based carboxylicacid esters (e.g., the “Span” surfactants, Fluka Chemika), includingsorbitan monolaurate (Span 20), sorbitan monopalmitate (Span 40),sorbitan monostearate (Span 60) and sorbitan monooleate (Span 80), andperfluorinated polyethers (e.g., DuPont Krytox 157 FSL, FSM, and/orFSH). Other non-limiting examples of non-ionic surfactants which may beused include polyoxyethylenated alkylphenols (for example, nonyl-,p-dodecyl-, and dinonylphenyls), polyoxyethylenated straight chainalcohols, polyoxyethylenated polyoxypropylene glycols,polyoxyethylenated mercaptans, long chain carboxylic acid esters (forexample, glyceryl and polyglycerl esters of natural fatty acids,propylene glycol, sorbitol, polyoxyethylenated sorbitol esters,polyoxyethylene glycol esters, etc.) and alkanolamines (e.g.,diethanolamine fatty acid condensates and isopropanolamine-fatty acidcondensates).

In certain embodiments, the carrier fluid may be caused to flow throughthe outlet channel so that the surfactant in the carrier fluid coats thechannel walls. In one embodiment, the fluorosurfactant can be preparedby reacting the perfluorinated polyether DuPont Krytox 157 FSL, FSM, orFSH with aqueous ammonium hydroxide in a volatile fluorinated solvent.The solvent and residual water and ammonia can be removed with a rotaryevaporator. The surfactant can then be dissolved (e.g., 2.5 wt %) in afluorinated oil (e.g., Flourinert (3M)), which then serves as thecarrier fluid.

Manipulating Droplet Size

The invention recognizes that in a fluidic circuit, changing thepressure exerted on the aqueous phase changes the flow rate of theimmiscible carrier fluid. Changing the flow rate of the immiscible fluidmanipulates the size of the droplet. Thus, adjusting pressure, whichchanges flow rate, adjusts droplet size. Pressure adjustments may bemade independently of each other such that the pressure exerted oil theaqueous phase in individual fluidic circuits can be adjusted to producedroplets of uniform size from the different fluidic circuits. In thismanner, droplets produced from different fluidic circuits travel at thesame velocity in a main channel and do not collide or coalesce in anunwanted manner. When the pressure is the variable parameter used forcontrol, there is coupling between the aqueous and immiscible carrierfluid (e.g., oil) channels in an individual circuit. Therefore, anychange to the aqueous pressure has an impact on the pressure at thenozzle and in turn affects the flow rate of the immiscible carrier fluid(IMF). For instance, increasing P_(Aq), decreases Q_(IMF) andvice-versa. Proper design of the resistances in both the aqueous andimmiscible carrier fluid channels controls the degree of coupling thatcan be expected when making a change to one or more of the inputpressures. This in turn controls the sensitivity of the change in dropvolume as a function of P_(A).

For comparison, the sensitivity of drop size to a change in flow rate iscompared using both a positive displacement pump and a pressure drivensystem. FIG. 3 is a graph showing droplet size sensitivity to changes inaqueous flow rate when using positive displacement pumping. FIG. 4 is agraph showing droplet size sensitivity to changes in aqueous flow ratewhen using pressure driven pumping. Oil was used as the immiscible fluidfor these comparisons. Using a similar chip with a similar circuit, apositive displacement pump yields a 10% change in drop volume whenchanging the flow rate by a factor of two. The pressure driven systemyields a 2% change in drop volume for every psi of change in P_(A). Ifthe pressure was doubled, a 60% change in drop size could be expectedwhen using the pressure driven system. Using a similar circuit pressuregives 6× better control over the droplet volume when the aqueous channelis adjusted.

In certain embodiments, multiple fluidic circuits are used to producedroplets that all flow into a main channel. Proper design of the fluidiccircuits, specifically by adjusting the fluidic resistance in both theaqueous and oil channels, controls the degree of influence thatadjustments to the aqueous pressure has on each of the circuits,resulting in all of the circuits producing droplets of the same size.Changes in droplet size as a result of changes in pressure and flow ratecan be modeled using the below calculations.

FIG. 5 shows a diagram of a single fluidic circuit for calculationpurposes. One of skill in the art will recognize that the calculationsshown herein may be applied to multiple fluidic circuits. (A) representsan immiscible carrier fluid channel, (B) represents an aqueous channel,(C) represents a junction of channels (A) and (B) where aqueous phaseand immiscible carrier fluid phase meet to form droplets of the aqueousphase surrounded by the immiscible carrier fluid, and (D) representsoutlet channel that receives the droplets. P_(A) represents the pressureof the immiscible carrier fluid in the immiscible carrier fluid channel.P_(B) represents the pressure of the aqueous fluid in the aqueous fluidchannel, P_(C) represents the pressure at the junction of channels (A)and (B). P_(A), P_(B), and P_(C) are all greater than 0, and P_(D) isequal to 0 because channel (D) is open to the atmosphere. Q_(AC)represents the flow rate of the immiscible fluid, Q_(BC) represents theflow rate of the aqueous fluid, and Q_(CD) represents the flow rate ofdroplets in channel (P). R_(AC) represents the fluidic resistance in theimmiscible carrier fluid channel, R_(BC) represents the fluidicresistance in the aqueous channel and R_(CD) represents the fluidicresistance in the (D) channel. Equations and expressions for Q_(AC) andQ_(BC) are as follows:

PA−PC=QAC(RAC)  Equation 1;

PB−PC=QBC(RBC)  Equation 2; and

PC=QCD(RCD)=(QAC+QBC)RCD  Equation 3.

Assuming that PA, PB, RAC, RBC, and RCD are known, then the threeunknowns are PC, QAC, and QBC. QAC and QBC can be solved for as follows:

$\begin{matrix}{{QAC} = {\frac{{{PA}({RBC})} + {\left( {{PA} - {PB}} \right){RCD}}}{{{RAC}({RBC})} + {{RCD}\left( {{RAC} + {RBC}} \right)}}\mspace{14mu} {and}}} & {{Equation}\mspace{14mu} 3} \\{{QBC} = {\frac{{{PB}({RAC})} + {\left( {{PA} - {PB}} \right){RCD}}}{{{RAC}({RBC})} + {{RCD}\left( {{RAC} + {RBC}} \right)}}.}} & {{Equation}\mspace{14mu} 4}\end{matrix}$

The sensitivities of the follow rates (Q) to changes in pressure (P) aredetermined oy obtaining partial derivatives of QAC and QBC with respectto PA and PB, which yields:

$\begin{matrix}{{\frac{\delta \; {QA}}{\delta \; {PA}} = \frac{\left( {{RBC} + {RCD}} \right)}{{{RAC}({RBC})} + {{RCD}\left( {{RAC} + {RBC}} \right)}}};} & {{Equation}\mspace{14mu} 5} \\{{\frac{\delta \; {QA}}{\delta \; {PB}} = {- \frac{RCD}{{{RAC}({RBC})} + {{RCD}\left( {{RAC} + {RBC}} \right)}}}};} & {{Equation}\mspace{14mu} 6} \\{{\frac{\delta \; {QB}}{\delta \; {PB}} = \frac{\left( {{RAC} + {RCD}} \right)}{{{RAC}({RBC})} + {{RCD}\left( {{RAC} + {RBC}} \right)}}};{and}} & {{Equation}\mspace{14mu} 7} \\{\frac{\delta \; {QB}}{\delta \; {PA}} = {{- \frac{RCD}{{{RAC}({RBC})} + {{RCD}\left( {{RAC} + {RBC}} \right)}}} = {\frac{\delta \; {QA}}{\delta \; {PB}}.}}} & {{Equation}\mspace{14mu} 8}\end{matrix}$

Assuming that P′A=PA+δPA then:

$\begin{matrix}{{\frac{Q^{\prime}A\; C}{QAC} = {1 + \frac{\left( {{RBC} + {RCD}} \right)\delta \; {PA}}{{{PA}({RBC})} + {\left( {{PA} - {PB}} \right){RCD}}}}};{and}} & {{Equation}\mspace{14mu} 9} \\{\frac{Q^{\prime}{BC}}{QBC} = {1 - {\frac{({RCD})\delta \; {PA}}{{{PB}({RAC})} - {\left( {{PA} - {PB}} \right){RCD}}}.}}} & {{Equation}\mspace{14mu} 10}\end{matrix}$

Similarly, assuming that P″B=PB+δPB then:

$\begin{matrix}{{\frac{Q^{''}A\; C}{QAC} = {1 - \frac{({RCD})\delta \; {PB}}{{{PA}({RBC})} + {\left( {{PA} - {PB}} \right){RCD}}}}};{and}} & {{Equation}\mspace{14mu} 11} \\{\frac{Q^{''}{BC}}{QBC} = {1 + {\frac{\left( {{RAC} + {RCD}} \right)\delta \; {PB}}{{{PB}({RAC})} - {\left( {{PA} - {PB}} \right){RCD}}}.}}} & {{Equation}\mspace{14mu} 12}\end{matrix}$

Substituting chip dPCR 1.3 specifics into the above and assuming PA≈PB,thus neglecting PA−PB containing terms yields:

$\begin{matrix}{{\frac{Q^{\prime}A}{QA} = {1 + {1.4\frac{\delta \; {PA}}{PA}}}};} & {{Equation}\mspace{14mu} 13} \\{{\frac{Q^{\prime}B}{QB} = {{- 0.45}\frac{\delta \; {PA}}{PA}}};} & {{Equation}\mspace{14mu} 14} \\{{\frac{Q^{''}A}{QA} = {1 - {0.36\frac{\delta \; {PB}}{PB}}}};{and}} & {{Equation}\mspace{14mu} 15} \\{\frac{Q^{''}B}{QB} = {1 + {3{\frac{\delta \; {PB}}{PB}.}}}} & {{Equation}\mspace{14mu} 16}\end{matrix}$

The results in FIG. 4 show that changing PA from 28 psi to 30 psiresults in Q_(BC) going from 577 μL/hr to 558 μL/hr, −3.3% change. Theabove model predicts a −3.1% change in QB₁ which is in agreement withthe actually results data.

In certain embodiments, the system may be configured such that thecircuits produce droplets of different size to allow for controlleddroplet coalescence in the main channel. The fluidic circuits arearranged and controlled to produce an interdigitation of droplets ofdifferent sizes flowing through a channel. Such an arrangement isdescribed for example in Link et al. (U.S. patent application numbers2008/0014589, 2008/0003142, and 2010/0137163) and European publicationnumber EP2047910 to Raindance Technologies Inc. Due to size variance,the smaller droplet will travel at a greater velocity than the largerdroplet and will ultimately collide with and coalesce with the largerdroplet to form a mixed droplet.

Another benefit of the added resistance in both channels next to thenozzle occurs during priming. Simultaneous arrival of both the aqueousand carrier liquids is difficult to produce reliably, if the carrierfluid enters the aqueous channel and travels all the way back into thefilter elements, the aqueous and carrier liquids begin to mix andemulsify before the nozzle. This mixing interference causes significantvariability in the size of the generated droplets. The added resistancenext to the nozzle eliminates the mixing interference by creating a pathof relatively high resistance without emulsifying features that are inthe filter. Therefore, if the carrier fluid arrives at the nozzle firstit will travel both into the aqueous resistor and towards the outlet ofthe chip. The outlet of the chip has a resistance that is much smallerthan the aqueous resistor and therefore the majority of the carrierfluid will flow in that direction. This gives the aqueous liquid time toreach the nozzle before the carrier fluid enters the filter feature.

Droplet Measurement

The volume of an individual droplet is measured using real-time imageanalysis. This in turn is fed back into a control loop where a knownprojected area is targeted and equal to a given droplet volume.Microfluidic chips are calibrated using a 3 point reference emulsion ofknow volumes to generate calibration curves for each channel. The ideais that the midpoint of the outline of a projected droplet image isalways the same regardless of the lighting. This demonstrated in FIG. 6,which is a drawing illustrating that the same volume drop is subject toextreme changes in the lighting but the midpoint is always the same.From left to right, the intensity of the lighting decreases but themidpoint of the outline is always the same. In contrast to determiningthe projected area of the inside of the drop, which is difficult due tochip and lighting imperfections and variability, or the outside of thedrop, which is also quite sensitive to lighting and chip imperfections,methods of the invention use the midpoint of the outline of a projecteddroplet image, which is always the same regardless of the lighting andchip imperfections. Using the midpoint “flattens out” the imperfectionsand is significantly less sensitive to outside influences on projecteddrop size. FIGS. 7A-C provides three graphs that demonstrate thedifferences in the droplet measuring techniques, and the projected arearequired to produce 5 pL drops when using the inside, outside andmidpoint of a droplet image. Finding both the outside and insideprojected area allows you calculate the outside and inside diameters.Calculating the average of the outside and inside diameters gives youthe midpoint diameter. From there an estimated projected area iscalculated from the midpoint diameter (See FIG. 8).

Nucleic Acid Target Molecules

One of skill in the art will recognize that methods and systems of theinvention are not limited to any particular type of sample, and methodsand systems of the invention may be used with any type of organic,inorganic, or biological molecule. In particular embodiments thedroplets include nucleic acids. Nucleic acid molecules includedeoxyribonucleic acid (DNA) and/or ribonucleic acid (RNA). Nucleic acidmolecules can be synthetic or derived from naturally occurring sources.In one embodiment, nucleic acid molecules are isolated from a biologicalsample containing a variety of other components, such as proteins,lipids and nontemplate nucleic acids. Nucleic acid template moleculescan be obtained from any cellular material obtained from an animalplant, bacterium, fungus, or any other cellular organism. In certainembodiments, the nucleic acid molecules are obtained from a single cell.Biological samples for use in the present invention include viralparticles or preparations. Nucleic acid molecules can be obtaineddirectly from an organism or from a biological sample obtained from anorganism, e.g., from blood, urine, cerebrospinal fluid, seminal fluid,saliva, sputum, stool and tissue. Any tissue or body fluid specimen maybe used as a source for nucleic acid for use in the invention. Nucleicacid molecules can also be isolated from cultured cells, such as aprimary cell culture or a cell line. The cells or tissues from whichtemplate nucleic acids are obtained can be infected with a virus orother intracellular pathogen. A sample can also be total RNA extractedfrom a biological specimen, a cDNA library, viral or genomic DNA.

Generally, nucleic acid can be extracted from a biological sample by avariety of techniques such as those described by Maniatis, et al.Molecular Cloning: A Laboratory Manual Cold Spring Harbor, N.Y., pp.280-281 (1982). Nucleic acid molecules may be single-stranded,double-stranded, or double-stranded with single-stranded regions (forexample, stem- and loopstructures).

Target Amplification

Methods of the invention further involve amplifying a target nucleicacid(s) in a droplet. Amplification refers to production of additionalcopies of a nucleic acid sequence and is generally carried out usingpolymerase chain reaction or other technologies well known in the art(e.g., Dieffenbach and Dveksler, PGR Primer, a Laboratory Manual, ColdSpring Harbor Press. Plainview, N.Y. [1995]). The amplification reactionmay be any amplification reaction known in the art that amplifiesnucleic acid molecules, such as polymerase chain reaction, nestedpolymerase chain reaction, polymerase chain reaction-single strandconformation polymorphism, ligase chain reaction (Barany F. (1991) PNAS88:189-193; Barany F. (1991) PCR Methods and Applications 1:5-16),ligase detection reaction (Barany F. (1991) PNAS 88:189-193), stranddisplacement amplification and restriction fragments lengthpolymorphism, transcription based amplification system, nucleic acidsequence-based amplification, rolling circle amplification, andhyper-branched rolling circle amplification.

In certain embodiments, the amplification reaction is the polymerasechain reaction. Polymerase chain reaction (PCR) refers to methods by K.B. Mullis (U.S. Pat. Nos. 4,683,195 and 4,683,202, hereby incorporatedby reference) for increasing concentration of a segment of a targetsequence in a mixture of genomic DNA without cloning or purification.

The process for amplifying the target sequence includes introducing anexcess of oligonucleotide primers to a DNA mixture containing a desiredtarget sequence, followed by a precise sequence of thermal cycling inthe presence of a DNA polymerase. The primers are complementary to theirrespective strands of the double stranded target sequence.

To effect amplification, primers are annealed to their complementarysequence within the target molecule. Following annealing, the primersare extended with a polymerase so as to form a new pair of complementarystrands. The steps of denaturation, primer annealing and polymeraseextension can be repeated many times (i.e., denaturation, annealing andextension constitute one cycle; there can be numerous cycles) to obtaina high concentration of an amplified segment of a desired targetsequence. The length of the amplified segment of the desired targetsequence is determined by relative positions of the primers with respectto each other, and therefore, this length is a controllable parameter.

Methods for performing PCR in droplets are shown for example in Link etal. (U.S. patent application numbers 2008/0014589, 2008/0003142, and2010/0137163), Anderson et al. (U.S. Pat. No. 7,041,481 and whichreissued as RE41,780) and European publication number EP2047910 toRaindance Technologies Inc. The content of each of which is incorporatedby reference herein in its entirety.

The sample droplet may be pre-mixed with a primer or primers and otherreagents for an amplification reaction, or the primer or primers andother reagents for an amplification reaction may be added to thedroplet. In some embodiments, fluidic circuits are controlled to producedroplets of different sizes to result in controlled merging of droplets.In those embodiments, sample droplets are created by segmenting thestarting sample and merging that droplet with a second set of dropletsincluding one or more primers for the target nucleic acid in order toproduce final droplets. The merging of droplets can be accomplishedusing, for example, one or more droplet merging techniques described forexample in Link et al. (U.S. patent application numbers 2008/0014589,2008/0003142, and 2010/0137163) and European publication numberEP2047910 to Raindance Technologies Inc.

In embodiments involving merging of droplets, two droplet formationmodules are used. A first droplet formation module produces the sampledroplets that on average contain a single target nucleic acid. A seconddroplet formation module produces droplets that contain reagents for aPCR reaction. Such droplets generally include Taq polymerase,deoxynucleotides of type A, C, G and T, magnesium chloride, and forwardand reverse primers, all suspended within an aqueous buffer. The seconddroplet also includes delectably labeled probes for detection of theamplified target nucleic acid, the details of which are discussed below.In embodiments that start with a pre-mix of sample and reagents for aPCR reaction, the pre-mix includes all of the above describedcomponents.

The droplet formation modules are arranged and controlled to produce aninterdigitation of sample droplets and PCR reagent droplets flowingthrough a channel. Such an arrangement is described for example in Linket al. (U.S. patent application numbers 2008/0014589, 2008/0003142, and2010/037163) and European publication number EP2047910 to RaindanceTechnologies Inc.

A sample droplet is then caused to merge with a PCR reagent droplet,producing a droplet that includes Taq polymerase, deoxynucleotides oftype A, C, G and T, magnesium chloride, forward and reverse primers,detectably labeled probes, and the target nucleic acid. Droplets may bemerged for example by: producing dielectrophoretic forces on thedroplets using electric field gradients and then controlling the forcesto cause the droplets to merge; producing droplets of different sizesthat thus travel at different velocities, which causes the droplets tomerge; and producing droplets having different viscosities that thustravel at different velocities, which causes the droplets to merge witheach other. Each of those techniques is further described in Link et al.(U.S. patent application numbers 2008/0014589, 2008/0003142, and2010/0137163) and European publication number EP2047910 to RaindanceTechnologies Inc. Further description of producing and controllingdielectrophoretic forces on droplets to cause the droplets to merge isdescribed in Link et al. (U.S. patent application number 2007/0003442)and European Patent Number EP2004316 to Raindance Technologies Inc.

Primers can be prepared by a variety of methods including but notlimited to cloning of appropriate sequences and direct chemicalsynthesis using methods well known in the art (Narang et al., MethodsEnzymol., 68:90 (1979); Brown et al., Methods Enzymol., 68:109 (1979)).Primers can also be obtained from commercial sources such as OperonTechnologies, Amersham Pharmacia Biotech, Sigma, and Life Technologies.The primers can have an identical melting temperature. The lengths ofthe primers can be extended or shortened at the 5′ end or the 3′ end toproduce primers with desired melting temperatures. Also, the annealingposition of each primer pair can be designed such that the sequence andlength of the primer pairs yield the desired melting temperature. Thesimplest equation for determining the melting temperature of primerssmaller than base pairs is the Wallace Rule (Td=2(A+T)+4(G+C)). Computerprograms can also be used to design primers, including but not limitedto Array Designer Software (Arrayit Inc.), Oligonucleotide ProbeSequence Design Software for Genetic Analysis (Olympus Optical Co.),NetPrimer, and DNAsis from Hitachi Software Engineering. The TM (meltingor annealing temperature) of each primer is calculated using softwareprograms such as Oligo Design, available from Invitrogen Corp.

Once final droplets have been produced, the droplets are thermal cycled,resulting in amplification of the target nucleic acid in each droplet.In certain embodiments, the droplets are flowed through a channel in aserpentine path between heating and cooling lines to amplify the nucleicacid in the droplet. The width and depth of the channel may be adjustedto set the residence time at each temperature, which can be controlledto anywhere between less than a second and minutes.

In certain embodiments, the three temperature zones are used for theamplification reaction. The three temperature zones are controlled toresult in denaturation of double stranded nucleic acid (high temperaturezone), annealing of primers (low temperature zones), and amplificationof single stranded nucleic acid to produce double stranded nucleic acids(intermediate temperature zones). The temperatures within these zonesfall within ranges well known in the art for conducting PCR reactions.See for example, Sambrook et al. (Molecular Cloning, A LaboratoryManual, 3rd edition, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y., 2001).

In certain embodiments, the three temperature zones are controlled tohave temperatures as follows: 95° C. (T_(H)), 55° C. (T_(L)), 72° C.(T_(M)). The prepared sample droplets flow through the channel at acontrolled rate. The sample droplets first pass the initial denaturationzone (T_(H)) before thermal cycling. The initial preheat is art extendedzone to ensure that nucleic acids within the sample droplet havedenatured successfully before thermal cycling. The requirement for apreheat zone and the length of denaturation time required is dependenton the chemistry being used in the reaction. The samples pass into thehigh temperature zone, of approximately 95° C., where the sample isfirst separated into single stranded DNA in a process calleddenaturation. The sample then flows to the low temperature, ofapproximately 55° C., where the hybridization process takes place,during which the primers anneal to the complementary sequences of thesample. Finally, as the sample flows through the third mediumtemperature, of approximately 72° C., the polymerase process occurs whenthe primers are extended along the single strand of DNA with athermostable enzyme.

The nucleic acids undergo the same thermal cycling and chemical reactionas the droplets passes through each thermal cycle as they flow throughthe channel. The total number of cycles in the device is easily alteredby an extension of thermal zones. The sample undergoes the same thermalcycling and chemical reaction as it passes through N amplificationcycles of the complete thermal device.

In other embodiments, the temperature zones are controlled to achievetwo individual temperature zones for a PCR reaction. In certainembodiments, the two temperature zones are controlled to havetemperatures as follows: 95° C. (T_(H)) and 60°10 C. (T_(L)). The sampledroplet optionally flows through an initial preheat zone before enteringthermal cycling. The preheat zone may be important for some chemistryfor activation and also to ensure that double stranded nucleic acid inthe droplets are fully denatured before the thermal cycling reactionbegins. In an exemplary embodiment, the preheat dwell length results inapproximately 10 minutes preheat of the droplets at the highertemperature.

The sample droplet continues into the high temperature zone, ofapproximately 95° C., where the sample is first separated into singlestranded DNA in a process called denaturation. The sample then flowsthrough the device to the low temperature zone, of approximately 60° C.,where the hybridization process takes place, during which the primersanneal to the complementary sequences of the sample. Finally thepolymerase process occurs when the primers are extended along the singlestrand of DNA with a thermostable enzyme. The sample undergoes the samethermal cycling and chemical reaction as it passes through each thermalcycle of the complete device. The total number of cycles in the deviceis easily altered by an extension of block length and tubing.

Target Detection

After amplification, droplets are flowed to a detection module fordetection of amplification products. The droplets may be individuallyanalyzed and detected using any methods known in the art, such asdetecting for the presence or amount of a reporter. Generally, thedetection module is in communication with one or more detectionapparatuses. The detection apparatuses can be optical or electricaldetectors or combinations thereof. Examples of suitable detectionapparatuses include optical waveguides, microscopes, diodes, lightstimulating devices, (e.g., lasers), photo multiplier tubes, andprocessors (e.g., computers and software), and combinations thereof,which cooperate to detect a signal representative of a characteristic,marker, or reporter, and to determine and direct the measurement or thesorting action at a sorting module. Further description of detectionmodules and methods of detecting amplification products in droplets areshown in Link et al. (U.S. patent application numbers 2008/0014589,2008/0003142, and 2010/0137163) and European publication numberEP2047910 to Raindance Technologies Inc.

In certain embodiments, amplified target are detected using detectablylabeled probes. In particular embodiments, the detectably labeled probesare optically labeled probes, such as fluorescently labeled probes.Examples of fluorescent labels include, but are not limited to, Attodyes, 4-acetamido-4′-isothiocyanatostilbene-2,2′disulfonic acid;acridine and derivatives: acridine, acridine isothiocyanate;5-(2′-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS);4-amino-N-[3-vinylsulfonyl)phenyl]naphthalimide-3,5 disulfonate;N-(4-anilino-1-naphthyl)maleimide; anthranilamide; BODIPY; BrilliantYellow; coumarin and derivatives; coumarin, 7-amino-4-methylcoumarin(AMC, Coumarin 120), 7-amino-4-trifluoromethylcouluarin (Coumaran 151);cyanine dyes; cyanosine; 4′,6-diaminidino-2-phenylindole (DAPI);5′5″-dibromopyrogallol-sulfonaphthalein (Bromopyrogallol Red);7-diethylamino-3-(4′-isothiocyanatophenyl)-4-methylcoumarin;diethylenetriamine pentaacetate;4,4′-diisothiocyanatodihydro-stilbene-2,2′-disulfonic acid;4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid;5-[dimethylamino]naphthalene-1-sulfonyl chloride (DNS, dansylchloride);4-dimethylaminophenylazophenyl-4′-isothiocyanate (DABITC); eosin andderivatives; eosin, eosin isothiocyanate, erythrosin and derivatives;erythrosin B, erythrosin, isothiocyanate; ethidium; fluorescein andderivatives; 5-carboxyfluorescein (FAM),5-(4,6-dichlortriazin-2-yl)aminofluorescein (DTAF),2′,7′-dimethoxy-4′5′-dichloro-6-carboxyfluorescein, fluorescein,fluorescein isothiocyanate, QFITC, (XRITC): fluorescamine; IR144;IR1446; Malachite Green isothiocyanate;4-methylumbelliferoneorthocresolphthalein; nitrotyrosine;pararosaniline: Phenol Red; B-phycoerythrin; o-phthaldialdehyde; pyreneand derivatives: pyrene, pyrene butyrate, succinimidyl 1-pyrene;butyrate quantum dots; Reactive Red 4 (Cibacron™ Brilliant Red 3B-A)rhodamine and derivatives: 6-carboxy-X-rhodamine (ROX),6-carboxyrhodamine (R6G), lissamine rhodamine B sulfonyl chloriderhodamine (Rhod), rhodamine B, rhodamine 123, rhodamine Xisothiocyanate, sulforhodamine B, sulforhodamine 101, sulfonyl chloridederivative of sulforhodamine 101 (Texas Red );N,N,N′,N′tetramethyl-6-carboxyrhodamine (TAMRA); tetramethyl rhodamine;tetramethyl rhodamine isothiocyanate (TRITC); riboflavin; rosolic acid;terbium chelate derivatives: Cy3; Cy5; Cy5.5; Cy7; IRD 700; IRD 800; LaJolta Blue; phthalo cyanine; and naphthalo cyanine. Preferredfluorescent labels are cyanine-3 and cyanine-5. Labels other thanfluorescent labels are contemplated by the invention, including otheroptically-detectable labels.

During amplification, fluorescent signal is generated in a TaqMan assayby the enzymatic degradation of the fluorescently labeled probe. Theprobe contains a dye and quencher that are maintained in close proximityto one another by being attached to the same probe. When in closeproximity, the dye is quenched by fluorescence resonance energy transferto the quencher.

Certain probes are designed that hybridize to the wild-type of thetarget, and other probes are designed that hybridize to a variant of thewild-type of the target. Probes that hybridize to the wild-type of thetarget have a different fluorophore attached than probes that hybridizeto a variant of the wild-type of the target. The probes that hybridizeto a variant of the wild-type of the target are designed to specificallyhybridize to a region in a PCR product that contains or is suspected tocontain a single nucleotide polymorphism or small insertion or deletion.

During the PCR amplification, the amplicon is denatured allowing theprobe and PCR primers to hybridize. The PCR primer is extended by Taqpolymerase replicating the alternative strand. During the replicationprocess the Taq polymerase encounters the probe which is also hybridizedto the same strand and degrades it. This releases the dye and quencherfrom the probe which are then allowed to move away from each other. Thiseliminates the FRET between the two, allowing the dye to release itsfluorescence. Through each cycle of cycling more fluorescence isreleased. The amount of fluorescence released depends on the efficiencyof the PCR reaction and also the kinetics of the probe hybridization. Ifthere is a single mismatch between the probe and the target sequence theprobe will not hybridize as efficiently and thus a fewer number ofprobes are degraded during each round of PCR and thus less fluorescentsignal is generated. This difference in fluorescence per droplet can bedetected and counted. The efficiency of hybridization can be affected bysuch things as probe concentration, probe ratios between competingprobes, and the number of mismatches present in the probe.

Droplet Sorting

Methods of the invention may further include sorting the droplets. Asorting module may be a junction of a channel where the flow of dropletscan change direction to enter one or more other channels, e.g., a branchchannel, depending on a signal received in connection with a dropletinterrogation in the detection module. Typically, a sorting module ismonitored and/or under the control of the detection module, andtherefore a sorting module may correspond to the detection module. Thesorting region is in communication with and is influenced by one or moresorting apparatuses.

A sorting apparatus includes techniques or control systems, e.g.,dielectric, electric, electro-osmotic, (micro-) valve, etc. A controlsystem can employ a variety of sorting techniques to change or directthe flow of molecules, cells, small molecules or particles into apredetermined branch channel. A branch channel is a channel that is incommunication with a sorting region and a main channel. The main channelcan communicate with two or more branch channels at the sorting moduleor branch point, forming, for example, a T-shape or a Y-shape. Othershapes and channel geometries may be used as desired. Typically, abranch channel receives droplets of interest as detected by thedetection module and sorted at the sorting module. A branch channel canhave an outlet module and/or terminate with a well or reservoir to allowcollection or disposal (collection module or waste module, respectively)of the molecules, cells, small molecules or particles. Alternatively, abranch channel may be in communication with other channels to permitadditional sorting.

A characteristic of a fluidic droplet may be sensed and/or determined insome fashion, for example, as described herein (e.g., fluorescence ofthe fluidic droplet may be determined), and, in response, an electricfield may be applied or removed from the fluidic droplet to direct thefluidic droplet to a particular region (e.g. a channel). In certainembodiments, a fluidic droplet is sorted or steered by inducing a dipolein the uncharged fluidic droplet (which may be initially charged oruncharged), and sorting or steering the droplet using an appliedelectric field. The electric field may be an AC field, a DC field, etc.For example, a channel containing fluidic droplets and carrier fluid,divides into first and second channels at a branch point. Generally, thefluidic droplet is uncharged. After the branch point, a first electrodeis positioned near the first channel, and a second electrode ispositioned near the second channel. A third electrode is positioned nearthe branch point of the first and second channels. A dipole is theninduced in the fluidic droplet using a combination of the electrodes.The combination of electrodes used determines which channel will receivethe flowing droplet. Thus, by applying the proper electric field, thedroplets can be directed to either the first or second channel asdesired. Further description of droplet sorting is shown for example inLink et al. (U.S. patent application numbers 2008/0014589, 2008/0003142,and 2010/0137163) and European publication number EP2047910 to RaindanceTechnologies Inc.

Release from Droplets

Methods of the invention may further involve releasing the enzymes fromthe droplets for further analysis. Methods of releasing contents fromthe droplets are shown for example in Link et al. (U.S. patentapplication numbers 2008/0014589, 2008/0003142, and 2010/0137163) andEuropean publication number EP2047910 to Raindance Technologies Inc.

In certain embodiments, sample droplets are allowed to cream to the topof the carrier fluid. By way of non limiting example, the carrier fluidcan include a perfluorocarbon oil that can have one or more stabilizingsurfactants. The droplet rises to the top or separates from the carrierfluid by virtue of the density of the carrier fluid being greater thanthat of the aqueous phase that makes up the droplet. For example, theperfluorocarbon oil used in one embodiment of the methods of theinvention is 1.8, compared to the density of the aqueous phase of thedroplet, which is 1.0.

The creamed liquids are then placed onto a second carrier fluid whichcontains a destabilizing surfactant, such as a perfluorinated alcohol(e.g. 1H,1H,2H,2H-Perfluoro-1-octanol). The second carrier fluid canalso be a perfluorocarbon oil. Upon mixing, the aqueous droplets beginsto coalesce, and coalescence is completed by brief centrifugation at lowspeed (e.g., 1 minute at 2000 rpm in a microcentrifuge). The coalescedaqueous phase can now be removed and the further analyzed.

Microfluidic Chips

Micro fluidic chips for performing biological, chemical, and diagnosticassays are described in U.S. Published Patent Application No.US2008/0003142 and US2008/0014589, the content of each of which isincorporated by reference herein in its entirety. Such microfluidicdevices generally include at least one substrate having one or moremicrofluidic channels etched or molded into the substrate, and one ormore interconnects (fluid interface). The one or more interconnectscontain inlet modules that lead directly into the microfluidic channels,and serve to connect the microfluidic channel to a means for introducinga sample fluid to the channel. The one or more interconnects also serveto form a seal between the microfluidic substrate and the means forintroducing a sample. The one or more interconnects can be moldeddirectly into the microfluidic substrate, as one or more individualpieces, or as a single, monolithic self-aligning piece. The interconnectmay also be a separate piece and the entire assembly (the manifold,microfluidic chip, and interconnect) can be modular as well. Anexemplary interconnect is shown in FIG. 9, which shows the interconnectwith immiscible fluid port 901 and aqueous fluid port 902. FIG. 10 showsthe interconnect integrated with a manifold having oil reservoir 1003and a microfluidic chip thereunder. FIG. 11 shows the interconnect 1104integrated with a microfluidic chip 1105 with the manifold (not shown)removed.

Microfluidic chips according to the invention include a substratedefining at least one internal channel and at least one port in fluidcommunication with the channels. In one particular embodiment, a chip ofthe invention includes a top plate adhered to a bottom plate to form thesubstrate with the channel(s) and port(s). The top plate can include theport(s), and the bottom plate can include the channel(s), such that whenthese two plates are brought together and adhered to each other thecombination forms the substrate with the channel(s) and the port(s). Themicrofluidic chip can be injection molded from a variety of materials.Preferably the microfluidic chip is injection molded using a cyclicolefin copolymer (COC) or cyclic olefin polymer (COP) or blend of COCand COP.

Chips of the invention include one or more fluidic circuits. Eachcircuit including a sample fluid channel, an immiscible carrier fluidchannel, and an outlet channel. The channels of each circuit areconfigured such that they meet at a junction so that droplets of aqueousfluid surrounded by carrier fluid are formed at the junction an flowinto the outlet channel. The outlet channel of each circuit is connectedto a main channel that receives all of the droplets from the differentfluidic circuits and flows the droplets to different modules in the chipfor analysis. In certain embodiments, each fluid circuit carries adifferent aqueous sample fluid in order to produce different sampledroplets. In other embodiments, the fluidic circuits all carry the sameaqueous sample fluid, and thus produce the same sample droplets.

A pressure source, optionally coupled to electronic pressure regulators,is used to pump fluids through multiple microfluidic channels inparallel. Multiple pressure regulators control the aqueous inputs. Theimmiscible carrier fluid input is under gain control for all channelssimultaneously. In this configuration, them is independent control ofindividual circuits to adjust projected area to obtain a target dropletvolume. Droplet volume is measured either relatively or absolutely(depending on the application) via real-time image analysis. Properdesign of the microfluidic circuits is required to obtain sensitive andprecise control of the droplet volume in all channels.

Pressure driven flow allows for the replacement of expensive mechanicalpans with inexpensive pneumatic control products. Pressure driven flowis instantaneous and pulse-free. Taking advantage of circuits inparallel, constant pressure driven flow instantly adjusts to changes inresistance in any and all channels without affecting any of the otherchannels.

Any pressure sources known in the art may be used with chips of theinvention. In certain embodiments, the pressure source is coupled toelectronic regulators. When coupled to an electronic regulator, thepressure source may be an external compressor with a reservoir forpumping compressed nitrogen, argon or air. In embodiments that do notused electronic regulators, an internal air cylinder with a linearactuator is applied.

The regulators should be of a type capable of regulating gas pressurefrom about 0 to about 5 atm in 100 evenly spaced increments (0-10 V,step=0.1 V). Each aqueous input is independently driven and controlledby a separate pressure regulator. The immiscible fluid lines arecontrolled in a gain control fashion, where one regulator is used todrive and control the flow of immiscible fluid through the entiresystem.

INCORPORATION BY REFERENCE

References and citations to other documents, such as patents, patentapplications, patent publications, journals, books, papers, webcontents, have been made throughout this disclosure. All such documentsare hereby incorporated herein by reference in their entirety for allpurposes.

EQUIVALENTS

The invention may be embodied in other specific forms without departingfrom the spirit or essential characteristics thereof. The foregoingembodiments are therefore to be considered in all respects illustrativerather than limiting on the invention described herein.

1-17. (canceled)
 18. A system for forming droplets, the systemcomprising: a microfluidic substrate comprising a sample channel and acarrier fluid channel that connects to the sample channel at a junction;a manifold in fluid communication with the microfluidic substrate, themanifold configured to deliver an aqueous fluid to the sample channeland an immiscible carrier fluid to the carrier channel to thereby formdroplets of aqueous fluid surrounded by carrier fluid at the junction;and a pump in fluid communication with the microfluidic substrate, thepump configured to regulate pressure of the aqueous fluid at thejunction.
 19. The system of claim 18, further comprising a fluidinterface comprising a plurality of interconnects each aligned with andforming a seal with an inlet of one of the plurality of microfluidicchannels.
 20. The system of claim 19, wherein the manifold is in fluidcommunication with the microfluidic substrate via the fluid interface.21. The system of claim 18, wherein the microfluidic substrate comprisesa top plate and a bottom plate.
 22. The system of claim 18, wherein thechannels are etched into one or both of the top plate and the bottomplate.
 23. The system of claim 18, further comprising an outlet channeldownstream of the junction.
 24. The system of claim 18, wherein the pumpis coupled to an electronic pressure regulator.
 25. The system of claim18, wherein the pump is an external compressor comprising a reservoir ofnitrogen, argon, or air.
 26. The system of claim 18, wherein the pump isan internal air cylinder with a linear actuator.
 27. The system of claim18, wherein the pressure of the carrier fluid is regulated by automaticgain control.
 28. The system of claim 18, further comprising an imagerconfigured to analyze droplet volume in real time.
 29. The system ofclaim 18, wherein the microfluidic substrate comprises a plurality offluidic circuits, each comprising sample channel.
 30. The system ofclaim 18, wherein each fluidic circuit is controlled by a separate pump.31. The system of claim 30, wherein the fluid interface is molded ontothe microfluidic substrate.